Plague is a zoonosis that is present in wild rodent populations worldwide and is transmitted primarily by fleas. Yersinia pestis, the plague bacillus, is unique among the enteric group of gram-negative bacteria in having adopted an arthropod-borne route of transmission. Y. pestis has evolved in such a way as to be transmitted during the brief encounter between a feeding flea and a host. A transmissible infection primarily depends on the ability of Y. pestis to grow in the flea as a biofilm that is embedded in a complex extracellular matrix. Bacteria in the biofilm phenotype are deposited into the dermis together with flea saliva, elements which cannot be satisfactorily mimicked by needle-injection of Y. pestis from laboratory cultures. The objective of this project is to identify and determine the function of Y. pestis genes that mediate flea-borne transmission and the initial encounter with the host innate immune system at the infection site in the skin. We study the interaction of Y. pestis with its insect vector by using an artificial feeding apparatus to infect fleas with uniform doses of wild-type or specific Y. pestis mutants. We seek to identify Y. pestis genes that are required for the bacteria to infect the flea midgut and to produce a biofilm that blocks the flea foregut and that is required for efficient transmission. The strategy entails first identifying bacterial genes that are differentially expressed in the flea by gene expression analysis and other techniques. Specific mutations are then introduced into these genes, and the mutants tested for their ability to infect and block the flea vector. Identification of such transmission factors allows further studies into the molecular mechanisms of the bacterial infection of the flea vector. Detailed understanding of the interaction with the insect host may lead to novel strategies to interrupt the transmission cycle. Our studies of flea vector competence and vectorial capacity will be useful to develop more realistic mathematical modeling of the epidemiology of plague transmission and the conditions that lead to plague epizootics. During FY2016, we published a study on the vector competence of the cat flea Ctenocephalides felis. C. felis is generally considered to be a poor vector, but the reasons for its relative inefficiency were unknown. Our data demonstrate that when infected cat fleas are restricted to a feeding pattern typical of X. cheopis, Y. pestis can survive, multiply, and block the digestive tract. However, cat fleas will feed much more frequently if given the opportunity, and this feeding pattern significantly decreased vector competence in our experiments. Variation in foregut anatomical features and physiology, in addition to frequency and duration of feeding, may partially explain differences in vector competence between flea species. C. felis is a common domestic and peridomestic flea in parts of Africa and China where plague is endemic. Since it feeds on a variety of hosts, including humans and wild rodent reservoirs of Y. pestis, it has been considered as a vector that could be responsible for transmitting Y. pestis to humans. Our work suggests that the most likely scenario for C. felis to become infective would be after its host dies of septicemic plague and their normal feeding schedule is disrupted, giving Y. pestis an opportunity to establish a transmissible infection. Thus, feeding behavior, differences in host preference, and foregut anatomy, rather than an intrinsic resistance to infection, likely limits transmission of Y. pestis by the cat flea. In collaboration with the Genomics Unit of the RML Research Technologies Branch, we are examining and characterizing the transcriptomes of Y. pestis, Y. pseudotuberculosis, and our biofilm-producing Y. pseudotuberculosis mutant during growth in vitro and during infection of the flea, with the goal of identifying genes and gene regulatory pathways that are important for flea-borne transmission. The in vivo and in vitro transcriptomic comparisons between these three strains are designed to broadly identify candidate components of biofilm regulatory pathways and other genes important for the recent evolutionary adaptation to flea-borne transmission. Significant differences in the expression of orthologous genes in the flea might be indicative of evolutionary changes in gene regulatory pathways. These results will be published in FY2017. We developed a new model system incorporating standardized experimental conditions and viability controls to more reliably compare the infection and transmission dynamics of different flea vectors. We used this system to compare the relative efficiency of transmission by Xenopsylla cheopis, a rat flea known to be an efficient plague vector, and Oropsylla montana, a North American ground squirrel flea that has been considered to be a poor vector. We found that both X. cheopis and O. montana transmit Y. pestis equivalently, resolving a long-standing uncertainty concerning the vector competence of O. montana. These results will be published in FY2017. We developed and evaluated new experimental systems to maintain and monitor infection status and transmission efficiency of individual fleas at different times after infection. The data will be used to estimate values for important parameters such as the probability of flea vectors developing a transmissible infection after feeding on a bacteremic host and the transmission efficiency during a four-week period after infection. Limited data are available are currently available for these values, which are needed for understanding plague epidemiology. We will use these values in mathematical models to better understand conditions that give rise to periodic plague epizootics.